Generation of distinct neuronal subtypes is a prerequisite for formation of a functional nervous system. For example, generation of rods and the three subtypes of cone cells in the mammalian retina are essential for receiving environmental light cues. Abnormal photoreceptor cell fate specification is frequently associated with retinal congenital and degenerative diseases and studies in the last decades have identified numerous photoreceptor cell fate determinant genes. Mutations in these genes are often linked to retinal developmental disorders and diseases. However, the molecular mechanisms underlying how these genes specify distinct photoreceptor subtypes and how they are regulated remains incomplete. This proposal is focused on understanding the regulatory mechanisms that determine a mutually exclusive binary choice between two photoreceptor cell fates using the fruit fly, Drosophila melanogaster. The conservation of the regulatory genes determining cell fate of photoreceptors between mammals and flies is remarkable. In mammals the choice between rods or cones is determined by the interplay between the cone-rod homeobox (Crx) protein and the neural retina-specific leucine zipper (NRL) protein. The Drosophila orthologues of these genes, Otd and Tj, regulate the choice of a specific photoreceptor between expressing one of two photo-pigments, rhodopsin 5 (Rh5) or rhodopsin 6 (Rh6). Our recent studies revealed that in Drosophila photoreceptors, the function of these genes is regulated by the Hippo signaling pathway, a signaling system that has also been shown to be highly conserved in regulating cell division and organ growth in both mammals and flies. Our preliminary studies have strongly suggested that multiple microRNAs are also involved in this photoreceptor subtype specification. microRNAs have been illustrated playing important regulatory roles in various developmental and disease processes, but their role in photoreceptor subtype fate specification is still unknown. In this proposal, we will use genetic and molecular approaches to elucidate how these microRNAs specify photoreceptor subtype fates by regulating conserved fate determinant genes and the photo-pigment genes. In addition, we will investigate how the biogenesis of these microRNAs is regulated by the newly identified Hippo signaling pathway. These studies will provide novel molecular mechanisms underlying neuronal subtype fate specification that will help understand the basis for a variety of developmental disorders and potentially aid in the design of strategies for directed differentiation of human pluripotent cells into specific cell types to replace the damaged cells in degenerative neuronal tissues.